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Isolation from cattle manure and characterization of Bacillus licheniformis NLRI-X33 Secreting Cellulase Kim Tae-Ill, J. D. Han1, B. S. Jeon1,C.B. Yang1, K.N. Kim1and M.K. Kim2 1 National Livestock Research Institute, Rural Development Administration, Suweon, 441-350, Korea 2 Division of applied Chemistry, school of Agricultural Biotechnology center for Plant Molecular Genetics and Breeding Research, College of Agriculture and Life science, Seoul National University, Suwon, 441-744, Korea ABSTRACT A bacterium producing extracellular cellulase was isolated from cow feces and has been identified as Bacillus sp.. The isolate, NLRI-X33 was shown to be similar to Bacillus licheniformis on the basis of morphological and biochemical properties as well as the composition of cellular fatty acids. When the isolate was cultured in CMC media at 37C for 24hrs, CMCase, FPase and Avicelase activity was 1.65 U/ml, 0.13 U/ml and 0.18 U/ml whereas -glucosidase activity was not detected. The optimum pH and temperature for enzyme induction was 7.5 and 50C. The maximum CMCase activity was observed at pH 7.5 and 75C. When crude supernatant was used for zymogram analysis, four major bands were detected on CMC-SDS-PAGE. Key Words : CMCase, Avicelase, -1,4-glucosidase, Bacillus licheniformis INTRODUCTION The function of cellulase is to hydrolyse cellulose by breaking the -1,4-glycosidic linkage in the major plant structural polysaccharide. There are three main types of enzyme found in cellulase system that has endoglucanase(CMCase, EC 3.2.1.4) which cleaves internal -1,4-glycosidic bonds, exoglucanase (Avicelase, EC 3.2.1.91) which releases cellobiose from the non-reducing end of cellulose, and cellobiase (-1,4-glycosidase, EC 3.2.1.2) which hydrolysis cellobiose to glucose (Wood and Bhat, 1998.). These enzymes work synergistically to hydrolyze cellulose to single carbohydrate (Yu et al, 1998). Applications of cellulase can be to improve the nutritional quality and digestibility of consumption and digestibility of ruminant feeds, to facilitate composting, to provide sugar syrups for human or animal consumption feeds, to provide sugar syrups for human or animal consumption and to supply fine chemical through fermentation (Beguin and Albert, 1985, Gilbert and Hazlewood, 1993). Bacillus sp. (Fumiyasu et al., 1985, Kim et al., 1997, Yoon and Jung, 1997), Pseudomonas sp.(Berghem et al, 1976, Wood and Kim, 1968), Clostridium sp. (Fagerstam and Pettersson, 1979) and Cellulomonas sp.(Chey et al, 1990, 1992) were known generally as cellulolytic bacteria. It has been revealed that bacterial cellulases were inferior to those of fungi for hydrolyzing cellulose but they show a tendency to be more heat stable and are easier for genetic work. In this paper, we describe the isolation and identification of a cellulase-producing bacterium from soil, compost and cow feces, and some properties of the crude CMCase. MATERIALS AND METHODS Isolation and Identification of Bacteria To isolate cellulolytic bacteria, the gathered soil, compost and cow feces were suspended in 0.1% Carboxymethyl cellulose(CMC medium viscosity, Sigma Chem. Co.), 1.5% bacto-tryptone, 0.5% bacto-soyton, 0.5% NaCl and 1.5% agar. After incubation at 37C for 24hours, the colonies were inoculated with needle onto CMC medium again. After growth, the plates were stained with 0.1% congo-red solution (Sigma Chem. Co.). When the stained plates were destained with 0.1M NaCl, the strains which formed a zone were selected as cellulolytic bacteria. General characteristics of the isolate were determined according to the Bergeys Manual of Systematic Bacteriology(Bergey’s 1986) and the Manual for General Bacteriology (Gerhardt et al, 1981). Methyl esters of cellular fatty acids of the isolate were analyzed by a gas liquid chromatography(HP 6890 GC, Microbial ID, USA) and microbial identification software. Crude Enzyme Preparation The isolated stains, grown in CMC agar, were transferred to CMC broth and cultured at 37C. After incubation for 24hours, cells were removed by centrifugation at 14,000rpm for 20min at 4C and the supernatant was precipitated by 80% ammonium sulfate saturation for overnight at 4C. The precipitate was dissolved with 0.1M Na-acetate buffer(pH 5.5). The precipitate was used as crude enzyme for the assay of enzyme activity. Assay Cellulolytic Enzyme Activity CMCase and Avicelase activity were assayed using 2% CMC and 2% Avicel as a substrate in 0.1M Na-acetate buffer(pH5.5) at 50C for 15min. FPase was assayed using Whatman No.1 Filter paper(16cm) as a substrate in 0.1M Na-acetate buffer(pH 5.5) at 50 C for 60min. The amount of reducing sugar produced from CMC, Avicel and Filter paper after reaction were determined by Somogyi-Nelson method(Wood and Bhat, 1998). One unit of enzyme activity was defined as the amount of enzyme which catalyzed the production of 1mol of glucose per min. -glucosidase assayed using 1mM p-nitrophenyl--D-glucopyranoside(PNPG, Sigma Chem. Co.) as a substate in 0.1M Na-acetate buffer(pH 5.5) at 50C. After reaction for 30min, 1M Na2CO3 and distilled water were added to stop the reaction, and liberated p-nitrophenol is measured at 430nm. One unit of enzyme activity is the amount of enzyme required to release 1 mol p-nitrophenol per min . Determination of Enzyme Properties To determine pH stability of CMCase, 100l of crude enzyme solution was added to 400l of 0.1M Na-acetate (pH45.5), 0.1M Na-phosphate (pH67), 0.1M Tris-HCl (pH7.59.0), and 0.1M Carbonate-bicarbonate buffers (pH9.511). After standing at 4C for 24hour, the residual CMCase activity was assayed using 200l of each solution in the same way as described above. The optimum temperature of CMCase activity was determined by assaying at various temperatures. The temperature effect on the stability of the enzyme was tested through the residual activity after reaction for 1 hour. Electrophoresis and Zymogram Electrophoresis and Zymogram was performed according to Laemmli(1970) and Park et al.(1997). SDS-PAGE was carried out in 12.5% polyacrylamide gel containing 0.1% CMC under constant 100 volts. After running , proteins were stained with Coomassie Brilliant blue R-250. Zymogram was carried out by staining the gel with 0.5% congo-red solution after removing SDS with 1% Triton X-100. The molecular weight of activity bands were estimated by plotting the log of the molecular weight of standard markers, activity bands vs. the relative mobility(Rf). RESULTS AND DISCUSSION Screening for Cellulase-producing bacterium In the first step of screening, about 150 colonies from soil, compost and cow feces were selected on CMC agar by staining with 0.1% congo-red. And then, they were tested if hemolysis occur on 5% sheep blood agar or not. Nine isolates without showing hemolysis were tested for their ability to hydrolyse CM-cellulose at 50C. Of them, X-33 strain is showing highest extracellular CMCase activity (Table 1). There is no correlation between diameter of clear zone and CMCase activity, corresponding to Ha et al(1992.) Table 1. Comparison of diameter of clear zone on the medium and CMCase activity Strain Source Clear CMCase Hemolysisb a Zone (mm) activity(U/ml) X-9 Cow feces 12 0.65 X-10 Cow feces 11 2.12 X-21 Cow feces 11 2.20 X-33 Cow feces 10 2.22 S-12 Soil 9 1.49 FT-16 Compost 11 0.69 T-6 Soil 10 0.41 T-20 Soil 9 0.99 T-24 Soil 9 1.29 aDiameter of clear zone after cultivation at 37C on the CMC agar medium bThe stains were inoculated on he medium containing 5% sheep blood, 5% beef heart, 1% Tryptone, 0.5% sodium chloride and 1.5% agar and cultured at 37C for 24hrus Identification of Cellulase-producing bacterium Morphological and physiological characteristics of isolate X-33 are shown in Table 2. X-33 is a Gram-positive, spore-forming, and rod-shaped bacterium. The cellular fatty acid composition of X-33 was similar to that of Bacillus licheniformis with a similarity of 0.772. Major cellular fatty acids of X-33 were branched chain fattty acids such as 40.65% of 15: 0 iso, 33.04% of 15: 0 Anteiso and 8.33% of 17: 0 anteiso (Table 3). From the results, the isolate was identified as Bacillus licheniformis and named Bacillus licheniformis NLRI-X33. Table 2. Biochemical and physiolagical characteri-stics of isolates NLRI-X33 Characteristics Result Gram stainig / Shape +, Rod Catalase + Oxidase Spore + Motility at 22C + Acid production from: Glucose + Mannitol + Lactose Maltose Saccharose + Esculin + Arabinose + Arginine + Cellobiose + Galactose Raffinose Salicin + Sorbitol + Sucrose + Trehalose + Table 3. Composition of the cellar fatty acids of the isolate X-33 Fatty acid contents(%) Fatty acid contents(%) 13:0 iso 0.21 14: 0 iso 0.78 14:0 0.41 15: 0 iso 40.65 15:0Anteriso 33.04 16:1w7calcohol 0.31 16:0iso 2.35 16:0wllc 0.38 16:0 2.23 16:02oh 0.18 iso 17: 1 w10c 1.15 17: 0 iso 8.63 17: 0 anteriso 0.29 18:0 0.26 Activity of Cellulolytic Enzymes on Various Substrates B. licheniformis NLRI-X33 was grown at 37C for 24hour in substate-containing medium, which is either Filter Paper No. 1, CM-celllose or Avicel, to test for cellulolytic activity with a substrate as the carbon source(Table 4). Maximum enzyme activity appeared in CMCase with 0.1% CM-cellulose. Activity of FPase and avicelase was detected whereas -glucosidase was not done at all. This result indicated that CM-cellulose was revealed as effective substate for celluloytic production. However, this result was not corresponding to the suggestion of Chey et al(1990) that avicelase was the most inducible enzyme in cellulolytic bacteria . Table 4. Comparison of activities of FPase, Avicelase and -glucosidase on three substrates Substrate Activity(U/ml) FPase CMCase avicease -glucosidase Filter Paper 0.18 0.88 0 0 CMC 0.13 1.65 0.18 0 Avicel 0.14 1.03 0 0 a Effect of initial pH and Temperature on Enzyme Production Initial pH and incubation temperature of CMC medium examined to improve enzyme production of B. licheniformis NLRI-X33. To determine effect of initial pH on enzyme production, pH of CMC medium was adjusted from 4.5 to 8 by adding 0.1N HCl and 0.1N NaOH to medium. Fig. 1 shows that the optimal initial pH for enzyme production was at pH 7.5. To determine the effect of incubation temperature on enzyme production , the medium was incubated at 3060C for 24hrs. Fig. 2 shows that the optimal temperature was at 50C. The temperature below 40C and above 60C did not support high enzyme production. To determine the optimal time for harvesting the culture to obtain maximum enzyme yield and the growth curve at the optimal condition for enzyme production, initial pH 7.5 and 50C, culture was harvested at intervals of 12 hour during incubation (Fig. 3.). The growth and the enzyme production increased rapidly during 0 to 36hrs but the growth kept in stationary phase at 48hrs whereas the enzyme production increased continuously. When cultured for 48hrs, the maximum quantity of CMCase and the growth were 1.5U/ml and 7.2 Log CFU/ml Relative activity(%) 100 80 60 40 20 0 4 5 6 7 8 9 Initial pH Fig. 1 Effect of initial pH on the CMCase production by B. licheniformis NLRI-X33. Relative activity (%) 100 80 60 40 20 0 30 Activity (units/mL) 0.1% Avicel, 0.1% CM-cellulose or Filter Paper No.1 (212cm) 50 60 6 0.5 5 12 24 36 48 60 4 84 72 Incubation time (hrs) Fig. 3 Time course of growth and CMCase production in a culture of B. licheniformis NLRI-X33. Viable cell count() and CMCase activity() in the culture medium (initial pH 7.5) Effects of Temperature and pH on CMCase Activity in Crude Extract The enzyme activity was assayed at temperature ranging 40-80C for 15min. Figure 4 shows that the optimum temperature for hydrolyzing CMcellulose was 65C. The crude enzyme stability was tested by various temperatures for 1 hour and then measured the remaining activity. This enzyme was stable at temperatures below 50C for 1 hour (about 10% loss). However, residual activity was 50% above 70C for 1 hour. The reported optimum temperature of the bacterial CMCase is 65C (Kim et al, 1997), and that of CMCase from Trichoderma sp. C-4 is 50C (Son et al,1997). To study the effect of pH on CMCase activity, released reducing sugar was measured after the crude enzyme was incubated with 2% CMcellulose in various buffers raging pH 4.011.0 at 50C for 15min. The crude enzyme was found to be most active in the range of pH 6.5-7.5 at 50C with above 90% of its activities(Fig.5). The crude enzyme effectively hydrolyzed 2% CMcellulose in the 0.1M Tris-HCl (pH7.5). To investigate the effect of pH on enzyme stability, samples of the crude enzyme incubated was assayed. Fig. 5 shows that the activities for hydrolysis of CMcellulose were stable between pH 7.5 and 8.0 The residual activities(%) are the values exchanged pH for the enzyme activity in 0.1M Tris-HCl (pH7.5) is settled to 100%. The optimal pH for the enzyme activity shows the pH range for enzyme stability of the CMCase-producing bacteria in the range of the pH 7.0-8.0. The reported pH stability of the bacterial CMCase is between pH 4.0 and 7.0 (Son et al, 1997), that of Bacillus stearothemophilus No.236 is between pH 6.0 and 7.0 (Kim et al ,1997). lati Incubation Temperature (¡É) Fig. 2 Effect of incubation temperature on CMCase Production of CMCase from B. licheniformis NLRI-X33. 1.0 0.0 0 Re 40 7 Log CFU/mL a 8 1.5 ve 100 80 60 40 ac 20 tivi 0 ty (% 30 40 50 60 Temperature (? ) 70 80 Fig. 4 Effect of temperature on activity and stability of the CMCase from B. licheniformis NLRI-X33. The enzyme activity was assayed at various temperature for 15min()and the residual activity was measured activity was measured after incubation of enzyme various temperature for 1hr() Relative activity(%) 100 80 60 40 20 0 4 5 6 7 8 9 10 11 pH Fig. 5 Effect of pH on activity and stability of the CMCase from B. licheniformis NLRI-33. The enzyme activity was assayed at various pH() and residual activity was measured activity was measured after incubation of the enzyme at various pH for 24hr(). Patterns of extracellular proteins and Zymogram Crude enzymes, collected at intervals of 12 hrs, were electrophoresed on 12.5% SDS-PAGE containing 0.1% CM-cellulose to study patterns of extracellular proteins and CMCase activity of Bacillus licheniformis NLRI-X33 on a gel. It appeared that patterns of proteins come to increase between 45 and 31kDa by passing incubation time (not shown). 4 major active bands, Cel 1, Cel 2, Cel 3 and 4, were detected by staining with 0.5% congo-red solution(Fig. 6). The molecular weight of the bacterial CMCase is 95kDa and 92kDa(Fumiyasu et al,1985, Kim et al,1997), and that of CMCase from Erwina sp. is 42,39,35,31 and 27kDa (Park et al,1997, Saarilahti et al,1990). 5.0 Cel 1 Log MW 4.8 Cel 2 Cel 3 4.6 Cel 4 4.4 4.2 4.0 0.0 0.2 0.4 0.6 0.8 1.0 Rf Fig 6. Molecular weights of Cel 1, Cel 2, Cel 3, and Cel 4 The molecular weight of Cel 1, Cel 2, Cel 3 and Cel 4 was estimated by platting the log of the molecular weights of standard markers, Cel 1, Cel 2, Cel 3 and 4 vs. the relative mobility(Rf) ACKNOWLEDGEMENT This work was supported by the 97 projects of Agricultural R&D promotion center, Ministry of Agriculture and Forestry, Korea REFERENCES Beguin, P. and J. P. Albert. 1994. The biological degradation of cellulose. FEMS Microbiol. Rev 13: 25-8. Bergey’s Mannual of Systematic Bacteriology. Vol 2. 1986. Williams and Wilkins press pp. 1104-1207. Berghem, L. E. R. L. G. Pettersson, and U. B. Axiofredriksson. 1976, The mechanism of enzymetic cellulose degredation Eur. J. Biochem. 61: 621-630. Chey, D. C. D. S. Kim, J. H. Yu, and D. H. Oh. 1990. Purification of cellulase produced from Cellulomona -s sp. YE-5. Kor. J. Appl. Microbiol. Biotechnol. 18: 376-168. Chey, D. C. D. S. Kim, J. H. Yu, and D. H. Oh. 1992. 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Kim, S. H. S. G. Cho, and Y. J. Choi. 1997. Purification and characterization of carboxymethyl cellulase from Bacillus stearothermophilus No. 236. J. Microbiol. Biotechnol. 7: 305-309. Laemmli, U. K. 1970. Cleavage of structure proein during the assembly of the head of bacteriophage T4. Natue (London) 227: 680-650.